Chrom

REVIEW
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A Decade of Development of
Chromogenic Culture Media for Clinical
Microbiology in an Era of Molecular
Diagnostics
John D. Perry
Downloaded from http://cmr.asm.org/ on May 4, 2018 by guest

Microbiology Department, Freeman Hospital, Newcastle upon Tyne, UK
SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 449 Published 25 January 2017

INTRODUCTION. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 450 Citation Perry JD. 2017. A decade of
CHROMOGENIC MEDIA FOR DETECTION OF SPECIFIC (NONENTERIC) PATHOGENS . 450 development of chromogenic culture media
Candida spp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 450 for clinical microbiology in an era of molecular
Pseudomonas aeruginosa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 451 diagnostics. Clin Microbiol Rev 30:449 – 479.
Staphylococcus aureus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 453 https://doi.org/10.1128/CMR.00097-16.
Streptococcus agalactiae (Group B Streptococcus) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 453
© Crown copyright 2017. The government of
Urinary Tract Pathogens. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 455
Australia, Canada, or the UK (“the Crown”) owns
CHROMOGENIC MEDIA FOR DETECTION OF ENTERIC PATHOGENS. . . . . . . . . . . . . . . . . . . . 456
the copyright interests of authors who are
Clostridium difficile . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 456
government employees. The Crown Copyright
Campylobacter spp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 457
is not transferable.
Salmonella spp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 458
Shigella spp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 458 Address correspondence to
Shiga Toxin-Producing Escherichia coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 459 john.perry@nuth.nhs.uk.
Vibrio spp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 460
Yersinia enterocolitica . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 460
CHROMOGENIC MEDIA FOR DETECTION OF ANTIMICROBIAL-RESISTANT
BACTERIA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 461
Methicillin-Resistant Staphylococcus aureus. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 461
Vancomycin-Resistant Enterococci . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 462
Extended-Spectrum-␤-Lactamase-Producing Enterobacteriaceae. . . . . . . . . . . . . . . . . . . . . . . . . 464
Carbapenemase-Producing Enterobacteriaceae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 465
Carbapenem-Resistant Acinetobacter spp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 467
IMPACT OF LABORATORY AUTOMATION ON THE USE OF CHROMOGENIC MEDIA . 468
CULTURE USING CHROMOGENIC MEDIA VERSUS MOLECULAR DIAGNOSTIC
METHODS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 469
CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 472
ACKNOWLEDGMENTS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 472
REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 473
AUTHOR BIO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 479
SUMMARY In the last 25 years, chromogenic culture media have found wide-
spread application in diagnostic clinical microbiology. In the last decade, the
range of media available to clinical laboratories has expanded greatly, allowing
specific detection of additional pathogens, including Pseudomonas aeruginosa, group B
streptococci, Clostridium difficile, Campylobacter spp., and Yersinia enterocolitica. New me-
dia have also been developed to screen for pathogens with acquired antimicrobial resis-
tance, including vancomycin-resistant enterococci, carbapenem-resistant Acinetobacter
spp., and Enterobacteriaceae with extended-spectrum ␤-lactamases and carbapenemases.
This review seeks to explore the utility of chromogenic media in clinical microbiology,
with particular attention given to media that have been commercialized in the last de-
cade. The impact of laboratory automation and complementary technologies such as
matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF
MS) is also assessed. Finally, the review also seeks to demarcate the role of chromogenic
media in an era of molecular diagnostics.
April 2017 Volume 30 Issue 2 Clinical Microbiology Reviews cmr.asm.org 449

Perry Clinical Microbiology Reviews
KEYWORDS carbapenemase-producing Enterobacteriaceae, chromogenic media,

methicillin-resistant Staphylococcus aureus, molecular methods
INTRODUCTION
C hromogenic media utilize synthetic chromogenic enzyme substrates in order to

specifically target pathogenic species (or groups of species) based on their enzyme
activity. Such enzyme activity is never completely species specific, necessitating the use
of complementary enzyme substrates and/or selective agents. The majority of chro-
mogenic media are therefore both selective and differential, accommodating the
inhibition of nontarget organisms (e.g., using antibiotics or other inhibitors) and
enabling target pathogens to grow as colored colonies due to their metabolism (usually
by hydrolysis) of one or more chromogenic enzyme substrates. The fact that only target
pathogens should generate colonies of a particular color reduces the number of
colonies that require investigation within a polymicrobial culture. Compared with the

use of conventional culture media, this often results in cost savings from reduced labor
time and reduced use of reagents, as fewer biochemical and/or serological confirmation
tests are required. This may contribute to quicker confirmation of pathogens and
reduce the overall time required to issue a report. In some cases, discrimination of
target pathogens from background flora due to generation of a specific color makes
pathogens less likely to be overlooked, thus improving rates of detection.
This review seeks to highlight the role of chromogenic culture media that have been
introduced since 2006 and to summarize evaluation data that have been published in
the last decade for preexisting applications. The review aims to clarify any advantages
or disadvantages compared with conventional methods and assess the relative merits
of culture using chromogenic media and those of competing molecular tests, such as
tests based on PCR techniques. The impact of laboratory automation, including matrix-
assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS)
and methods for automated colony detection, is also discussed. The review is confined
to solid media that have been used for the isolation of clinically important bacteria from
human samples in published evaluations. The majority of the studies considered here
are peer-reviewed articles published in journals since 2006 in English, with priority
given to studies that utilize patient samples rather than pure microbial strains. Con-
ference abstracts are cited sparingly and only when they offer additional insights. Since
2006, the array of chromogenic culture media available to clinical laboratories has
expanded, allowing the specific detection of many more pathogens of interest, such as
Clostridium difficile, Streptococcus agalactiae, Yersinia enterocolitica, Campylobacter spp.,
and Pseudomonas aeruginosa. In addition, an expanded range of media is now com-
mercially available to screen for bacteria with acquired mechanisms of antimicrobial
resistance, including vancomycin-resistant enterococci (VRE), carbapenem-resistant
Acinetobacter spp., and Enterobacteriaceae with extended-spectrum ␤-lactamases and
carbapenemases. Table 1 provides a timeline for the application of commercially
available chromogenic media to clinical diagnostics (1–17).
CHROMOGENIC MEDIA FOR DETECTION OF SPECIFIC (NONENTERIC)

PATHOGENS
Candida spp.
A chromogenic medium for the identification and differentiation of pathogenic
yeasts, CHROMagar Candida (CAC), was first reported in 1994 (2). As well as antibacterial
agents, the medium incorporates two chromogenic substrates for the detection of
␤-hexosaminidase activity and phosphatase activity (18). The medium affords specific
identification of Candida albicans/Candida dubliniensis, which form green colonies due
to production of ␤-hexosaminidase, and Candida tropicalis, which forms blue colonies
due to production of both enzymes. Other species of yeast form pink colonies due to
phosphatase activity alone or produce neither of these enzymes and grow as white
colonies. A range of commercially available chromogenic media has since been eval-
uated, including Albicans ID (19), CandiSelect (19), Candida ID (20), Candida diagnostic
April 2017 Volume 30 Issue 2 cmr.asm.org 450

Chromogenic Media for Clinical Microbiology Clinical Microbiology Reviews
TABLE 1 Timeline of the evolution of chromogenic culture media applied to clinical

diagnostics
Yr of first reported study
with clinical samples Targeted pathogen(s) Reference
1993 Salmonella spp. 1
1994 Candida spp. 2
1995 Urinary tract pathogens 3
2000 Staphylococcus aureus 4
Methicillin-resistant Staphylococcus aureus 5
2006 Streptococcus agalactiae 6
2007 Enterobacteriaceae with extended-spectrum 7
␤-lactamases
Vancomycin-resistant enterococci 8
2008 Enterobacteriaceae with carbapenemases 9
2009 Acinetobacter spp. 10
Pseudomonas aeruginosa 11
Shiga toxin-producing E. coli 12

2010 Clostridium difficile 13
2011 Campylobacter spp. 14
Vibrio spp. 15
2012 Shigella spp. 16
2013 Yersinia enterocolitica 17
agar (21), Pourmedia Vi Candida (22), Chromogenic Candida agar (23), Brilliance Can-
dida Agar (24) and HiCrome Candida differential agar (25). A common feature of these
media is a chromogenic substrate for ␤-hexosaminidase to discriminate C. albicans/C.
dubliniensis from other yeasts, and most include a second chromogenic substrate
(usually to detect phosphatase or ␤-glucosidase) to provide further discrimination
between species (18). The main advantage of such chromogenic agars is their ability to
detect mixed cultures of yeasts due to the fact that different species frequently form
colonies with different colors. Such mixtures of species may be indistinguishable and
remain undetected as mixtures on conventional agars such as Sabouraud agar plus
chloramphenicol (23, 26). This is important, as different species may have different
susceptibilities to antifungal agents. While C. albicans/C. dubliniensis are usually sus-
ceptible to antifungal agents, chromogenic media may help to detect species with a
higher likelihood of resistance to azoles and/or amphotericin B, including Candida
krusei, Candida glabrata, Candida rugosa and Candida inconspicua (27).
There have been relatively few comparisons of different chromogenic agars using
clinical specimens in the last decade. Ozcan et al. compared Oxoid Chromogenic
Candida agar (OCCA) with CHROMagar Candida (CAC) and Sabouraud chloramphenicol
agar (SCA) using 392 vaginal swabs. Yeasts were isolated from 161 samples, and 21
samples (13%) yielded a mixture of species on at least one medium (23). OCCA and CAC
showed comparable sensitivity (96.9% versus 97.5%, respectively) for detection of
positive samples, whereas the sensitivity of SCA was lower (91.9%). For the 21 poly-
fungal infections, 20 (95.2%) were detected using OCCA, compared with only 14
(66.7%) using CAC (P ⬍ 0.05). Sendid et al. compared CandiSelect 4 (CS4) with CAC
using 1,549 clinical samples from a wide variety of sites (28). A total of 502 samples
(32.4%) yielded one or more yeast species, including 37 samples (7.4%) that yielded
more than one species. The sensitivities of CS4 and CAC were very similar (92.1 and
91.1%, respectively), with no false-positive results. CS4 was superior to CAC for pre-
sumptive identification of C. glabrata (80 versus 75%) and C. krusei (92 versus 83%) but
was less effective for C. tropicalis (68 versus 76%).
Pseudomonas aeruginosa
P. aeruginosa is an important nosocomial pathogen and may also cause community-
acquired infections, particularly in individuals with underlying disease. For example, in
patients with cystic fibrosis, it is a common and important cause of respiratory tract
infection. Laine et al. reported the first chromogenic medium designed specifically for
the isolation of P. aeruginosa (PS-ID), which was subsequently commercialized as


FIG 1 Examples of chromogenic media for detection of specific pathogens that have been first reported
in the last decade. (a) Colony variants of Pseudomonas aeruginosa isolated from the sputum of a patient
with cystic fibrosis after 36 h of incubation on chromID P. aeruginosa (reprinted from reference 11 with
permission). (b) Dark blue colonies of Streptococcus agalactiae mixed with pink colonies of Enterococcus
faecalis after 18 h of incubation on StrepBSelect. (c) Typical colonies of Clostridium difficile after 48 h of
incubation on chromID C. difficile. (d) Red colonies of Campylobacter jejuni on CASA medium after 48 h
of incubation. (e) Mauve colonies of a pathogenic biovar of Yersinia enterocolitica among blue colonies of
background flora on CHROMagar Y. enterocolitica. (f) Red colonies of Acinetobacter baumannii isolated on
CHROMagar Acinetobacter (this medium has an optional supplement to select for carbapenem-resistant
strains). (Panels e and f are courtesy of CHROMagar, Paris, France; reproduced with permission.)
chromID Pseudomonas (11). The medium is notable as it is the first chromogenic

medium to utilize a chromogenic substrate for peptidase activity. This substrate,
␤-alanyl pentylresorufamine, is hydrolyzed by a ␤-alanyl aminopeptidase produced by
P. aeruginosa, resulting in the formation of purple colonies (29) (Fig. 1a). The medium
was evaluated with 100 sputum samples from patients with cystic fibrosis and com-
pared with Pseudomonas CN selective agar (CN). The recovery of P. aeruginosa was
equivalent on both media (95.2%), but the positive predictive value of PS-ID (98.3%)
was significantly higher than that of growth on CN (88.5%) for identification of P.
aeruginosa (P ⬍ 0.05). Other species of Gram-negative bacteria were occasionally

isolated as purple colonies on PS-ID, including Burkholderia cepacia complex (11). There
are no other reports of this medium with clinical samples; however, Weiser et al.
included chromID Pseudomonas in a comparison of five selective media that were
challenged with 50 isolates of P. aeruginosa and 90 isolates belonging to closely related
species (30). chromID Pseudomonas showed the highest specificity of the five media
tested, but the authors reported that its sensitivity (95%) was negatively impacted by
the large variation in color of P. aeruginosa colonies (including pink-brown and green,
possibly due to interference from natural pigments of P. aeruginosa). In conclusion,
there is a lack of any published studies with clinical samples that demonstrate a higher
recovery of P. aeruginosa using chromogenic media.
Staphylococcus aureus
S. aureus is one of the most frequent and important human pathogens and is
implicated in a range of infections, including superficial skin infections, abscesses,
bacteremia, and food poisoning. It is frequently found colonizing the nose, throat, and

skin without causing symptoms. The first chromogenic medium for the isolation of S.
aureus, CHROMagar S. aureus, was reported in 2000 (4) and utilized a phosphatase
substrate for detection of S. aureus as pink colonies (18). Since that first report, at least
two other media have been made commercially available and evaluated with clinical
samples, including S. aureus ID (31) (later commercialized as chromID S. aureus) and
SaSelect (32). An alternative approach is utilized in chromID S. aureus, which relies upon
production of ␣-glucosidase by S. aureus, resulting in the formation of green colonies.
Each of these media has been reported to show sensitivity that is equivalent to or
higher than that of conventional nonselective media (e.g., blood agar), and, due to the
incorporation of selective agents for the inhibition of nonstaphylococci, they are
particularly useful for specimens that yield a polymicrobial flora that includes Gram-
negative bacteria. They also have high specificity (⬎90%) for detection of S. aureus,
meaning that fewer confirmation tests are required when reading culture plates (4,
31–34). The sensitivity may be increased if incubation is extended to 48 h, particularly
for CHROMagar S. aureus (31, 33), but this is offset by a small decrease in specificity due
to other species forming colonies of the same color as S. aureus (31, 33, 34). As there
is only one published “head-to-head” comparison of these chromogenic media with
clinical samples (31), there are insufficient data to conclude whether any particular
chromogenic medium is better than another.
Streptococcus agalactiae (Group B Streptococcus)

Infections caused by group B streptococci (GBS) are a leading cause of morbidity
and mortality in newborn infants. Asymptomatic carriage of GBS in the maternal
genitourinary tract may lead to colonization of the neonate, and in a small proportion
of cases, this may lead to invasive disease. In an effort to reduce the burden of disease,
authorities in many countries recommend universal screening of all pregnant women
for vaginal/rectal colonization by GBS at 35 to 37 weeks of gestation (35). A widely used
standard procedure involves overnight incubation of samples (i.e., vaginal/rectal swabs)
in a selective enrichment broth followed by subculture onto blood-based culture media
for investigation of typical hemolytic colonies (35). Granada medium is also widely
used, and this medium allows GBS to grow as orange colonies under anaerobic
conditions due to formation of a natural pigment (36).
The first chromogenic medium for GBS (a prototype of chromID Strepto B) was
described in 2006 (6). The medium allows GBS to form red colonies based on produc-
tion of phosphatase. Other species either form colorless colonies or hydrolyze addi-
tional chromogenic substrates (for esterase and ␤-cellobiosidase enzymes) to produce
blue/green colonies (37). Since this first report, a large number of studies have
evaluated a range of chromogenic media for detection of GBS. Table 2 summarizes a
selection of eight of these studies (i.e., those with the largest number of positive
samples). A number of studies have compared chromogenic media against selective
blood-based agars (usually containing colistin and nalidixic acid) with or without the

TABLE 2 Summary of studies evaluating chromogenic media for the isolation of Streptococcus agalactiae from clinical samples
Positive
Sensitivity (%) Specificity (%) predictive value
Total no. of
at: at: (%) at:
Study authors, samples/no.
yr (reference) positive Swab type(s) Test medium(a)a 18–24 h 48 h 18–24 h 48 h 18–24 h 48 h
Smith et al., 200/83 Vaginal chromID Strepto B 67.5 67.5 100 100
2008 (38) CNA blood agar 57 57 89.7 89.7
Broth (CN-TH), chromID Strepto B 91.6 92.8 100 100
Broth (CN-TH), blood agar 88 89.2 88.9 89.7
Craven et al., 250/81 Vaginal, rectal chromID Strepto B 87.7 97.6

2010 (39) Neo/nali blood agar 79 97.6
Broth (CN-TH)/blood agar 91.4 100
Louie et al., 1,025/243 Vaginal, rectal CNA blood agar 82.7

2010 (41) Broth, CNA blood agar 92.2
Broth, StrepB Select 98.8 99.2

Joubrel et al., 141/88 Vaginal Granada 96.5 100
2014 (46) Brilliance GBS 94.3 96.2
StrepB Select 97.7 91
chromID Strepto B 92 92 98.7
Poisson et al., 528/60 Vaginal, others chromID Strepto B 71.7 90 100 85.7
2010 (47) Granada 61.7 88.3 100 100
Blood agar 46.7 66.7 82.4 88.9
Poisson et al., 285/84 Vaginal, rectal Broth (CN-TH), CHROMagar StrepB 79 92 96 95

2011 (42) Broth (CN-TH), CNA blood agar 82 92
Broth (CN-TH), blood agar 40 58 92 91
Kwatra et al., 260/92 Vaginal, rectal CHROMagar StrepB 85.9 88

2013 (43) CNA blood agar 70.7 80.9
Broth (GN-TH), blood agar 55.4 78.4
Morita et al., 1,425/319 Vaginal, rectal Broth (CN-TH), chromID Strepto B 99.7
2014 (37) Broth (CN-TH), blood agar 93.7
aCNAblood agar, blood agar supplemented with colistin and nalidixic acid; broth (CN-TH), Todd-Hewitt broth supplemented with colistin and nalidixic acid; neo/nali
blood agar, blood agar supplemented with neomycin and nalidixic acid; broth (GN-TH), Todd-Hewitt broth supplemented with gentamicin and nalidixic acid.
use of a selective enrichment broth (38–44). When both types of media were tested
under the same conditions, chromogenic media showed a higher sensitivity than
selective blood agars in all of these studies. Most studies show that the sensitivity of any
culture medium for detection of GBS may be substantially improved by use of an
enrichment broth (38, 39, 41, 44, 45). Chromogenic media have a potential advantage
over Granada medium, as they do not require anaerobic incubation and have the ability
to detect nonhemolytic strains of GBS that typically fail to produce pigment on Granada
medium. Such strains are thought to account for up to 5% of invasive infections (46).
Despite this, several studies have compared Granada medium with chromogenic agars
(6, 40, 45–48), and overall there is no clear advantage of either in terms of sensitivity.
Moreover, Granada medium invariably demonstrates 100% specificity and is arguably
the only medium that can be used without the need to confirm the identification of
suspect colonies of GBS (46).
Only a few studies have compared the performance of different chromogenic media
for GBS in a head-to-head evaluation using clinical samples (46, 48, 49). No statistically
significant advantage was found for any of the media tested, and sensitivity is likely to
have been underestimated because they were not used in conjunction with an enrich-
ment broth (38, 44, 45). Most studies conclude that chromogenic media for GBS are
highly convenient tools that offer an increased sensitivity and specificity over conven-
tional blood-based media. Further large studies would be needed to establish the
superiority of any particular chromogenic medium, and the use of an enrichment broth
should ideally be included in such studies.

Urinary Tract Pathogens

The first report of a chromogenic medium for diagnosis of urinary tract infections
described an evaluation of CPS ID2 in 1995 (3). This medium exploited a substrate for
␤-glucuronidase to allow the specific identification of the most common urinary pathogen,
Escherichia coli, as pink or red colonies. An additional substrate for ␤-glucosidase allows
detection of enterococci as small green colonies and the Klebsiella-Enterobacter-Serratia
(KES) group as larger green colonies. Finally, the inclusion of tryptophan and iron salts
allows Proteeae (Proteus-Providencia-Morganella) to form brown colonies due to deaminase
activity (50).
A range of other media has since been commercialized and evaluated with clinical
samples, including CHROMagar Orientation (51), UriSelect medium (52), Rainbow Agar
UTI medium (52), Chromogenic UTI medium (52), USA agar (53), Harlequin CLED (54),
and Urichrom agar (55). A number of these media, including CHROMagar Orientation,
UriSelect medium, and Chromogenic UTI medium, utilize a substrate for ␤-galactosidase

for detection of E. coli (18). This allows the direct identification of a higher proportion
of E. coli isolates, as approximately 99% of E. coli isolates produce ␤-galactosidase,
compared with approximately 94% that produce ␤-glucuronidase (18). However, this
advantage is offset by a small decrease in specificity due to the misidentification of a
proportion of Citrobacter spp. as E. coli in some reports (56, 57). This small decrease in
specificity can be largely eliminated by inclusion of a spot indole test, but this is
laborious as it needs be applied to all colonies resembling E. coli (56).
In some studies, chromogenic media have been shown to provide a superior
differentiation of mixed cultures due to the fact that different species may generate
colonies with different colors and may not be easily differentiated on conventional
agars. This can assist in the recognition of urine samples that may be contaminated,
particularly compared with culture on cystine-lactose-electrolyte-deficient (CLED) agar
(54, 56). However, this advantage is not apparent in other studies (58, 59). A consistent
advantage of chromogenic media is their ability to identify E. coli and provide identi-
fication of other groups of species (KES and Proteeae). Several groups have shown how
that can contribute to a decrease in workload for species identification and/or to cost
savings to the laboratory (60–62); however, this may have no significant impact on the
overall time taken to generate a final report (62).
Chromogenic media designed for detection of urinary tract infections are unique
among chromogenic media as they do not contain antimicrobials as selective agents in
order to cultivate as many species as possible. They can therefore potentially be used
as single media for the culture of urine samples. In one of the largest reported studies,
Aspevall et al. (58) evaluated four chromogenic media, i.e., Chromogenic UTI medium,
CHROMagar Orientation from two commercial sources, and CPS ID2, alongside culture
on CLED, blood agar, and MacConkey agar using 1,200 urine samples. Although
incubation was extended for up to 48 h, this had a minimal impact on any of the test
media. A total of 420 isolates deemed to be potentially significant were recovered from
379 urine samples at a count of ⱖ104 CFU/ml. A total of 96% of these isolates were
recovered on blood agar and also on CLED agar. The four chromogenic media recov-
ered between 92 and 96% of isolates. The authors concluded that any of the chromo-
genic media studied could be used as a single medium for the isolation of uropatho-
gens. The authors also reported that mixed urethral flora was easier to detect on blood
agar due to better growth of fastidious species such as corynebacteria and alpha-
hemolytic streptococci. They therefore advocated retaining blood agar as part of the
urine culture workup, as isolation and discrimination of different Gram-positive bacte-
rial species were found to be much easier with this medium. This has been noted by
others; for example, Yarbrough et al. (62) noted the recovery of a smaller amount of
periurethral flora (including lactobacilli and group B streptococci) on chromID CPS Elite
than with culture on blood agar. This resulted in fewer reports of contaminated or
insignificant growth. The authors concluded that chromID CPS Elite agar may be a

feasible alternative to conventional media for isolation and identification of most

common uropathogens in urine specimens.
Some brands of media have been subject to incremental improvements over the years
and new generations of media have been commercialized. In the last decade, four pub-
lished studies have compared either UriSelect 4 or CHROMagar Orientation with different
generations of CPS media (CPS ID3, CPS ID4, and chromID CPS Elite). The studies revealed
only minor differences in specificity between these media, with sensitivity reported to be
broadly equivalent (57, 59, 63, 64). Data are lacking on the effectiveness of chromogenic
media for recovery of some fastidious Gram-positive bacteria, some of which (e.g., Aero-
coccus urinae) have been implicated as human pathogens (63, 65).
CHROMOGENIC MEDIA FOR DETECTION OF ENTERIC PATHOGENS

Clostridium difficile
Over the last decade there has been renewed interest in the culture of stool samples

for the isolation of C. difficile. One reason is the emergence of so-called hypervirulent
strains that cause outbreaks of C. difficile infection (CDI) that are associated with an
increased severity of disease and significant mortality (66). In order to track the spread
of such strains, it is usually necessary to isolate them by culture and perform molecular
typing. Culture also affords a very high sensitivity for detection of C. difficile and may
therefore be useful in the diagnosis of CDI. In one large 7-year study, toxigenic culture
resulted in the diagnosis of 355 cases of CDI that would have been missed using the
fecal cytotoxin assay alone (67). For these reasons, isolation of C. difficile followed by
demonstration of toxin or toxin genes (“cytotoxigenic culture”) is accepted by many as
a “gold standard” for diagnosis of CDI (68).
The first chromogenic culture medium (IDCd) for isolation of C. difficile was reported
in 2010 (13). The principle was based upon the ability of C. difficile to generate black
colonies due to expression of ␤-glucosidase activity resulting in the hydrolysis of a
chromogenic substrate (Fig. 1c). The authors reported that IDCd offered effective
isolation of C. difficile within only 24 h of incubation with or without the use of alcohol
shock treatment, in contrast to other selective media. IDCd was subsequently commer-
cialized and marketed as chromID C. difficile. Since this first report, at least six published
articles have reported evaluation data for chromID C. difficile in comparison with other
media (69–74). Five of these studies are summarized in Table 3. In all cases, chromID C.
difficile showed a sensitivity superior to that of comparator media and resulted in
greater inhibition of other flora. In three of these studies, there was evidence that
incubation of chromID C. difficile for 48 h improved the sensitivity of the medium,
particularly for clinical samples with a low burden of C. difficile. In a further study,
chromID C. difficile and Oxoid Clostridium difficile selective agar (CCFA) were used for
the culture of 686 stool samples from 508 patients in four hospitals in Hong Kong, with
incubation of both media for up to 72 h (74). C. difficile was isolated from 118 stool
samples using chromID C. difficile, compared with 70 stool samples using CCFA (P ⬍
0.001); however, the overall sensitivity of the two media was not reported.
The high sensitivity afforded by chromID C. difficile is due to a combination of high
selectivity against unwanted bacteria and a strong propensity of the medium to
stimulate germination of spores (13). These attributes were exploited by Hill et al., who
demonstrated the superior sensitivity of chromID C. difficile for recovery of C. difficile
from environmental surfaces (75). In a study with 496 samples from the hospital
environment, the sensitivity of chromID C. difficile was 87.6%, compared with a
sensitivity of 26.6% for cefoxitin-cycloserine-egg yolk agar plus lysozyme, a medium
that has also been recommended for environmental screening (P ⬍ 0.0001) (76).
Despite the high sensitivity of chromID C. difficile, the medium also has some
limitations. The chromogenic reaction is not specific for C. difficile, and black colonies
may be produced by other anaerobic species, most notably Clostridium hathewayi (a
species previously classified as Clostridium clostridioforme) (74). Colonies recovered on
chromID C. difficile therefore require identification, and this can be readily achieved
using MALDI-TOF MS (74). Alternatively, Park et al. proposed the use of a Gram stain

TABLE 3 Summary of studies comparing chromID C. difficile with other culture media for
isolation of C. difficile from stool samples.
Sensitivity
(%) at:
Study authors, yr Total no. of Sample
(reference) samples/no. positive treatment Test mediuma 24 h 48 h
Eckert et al., 2013 (70) 406/54 None chromID C. difficile 74.1 87
TCCA 85.2
CLO 70.4
Carson et al., 2013 (69) 50/47 None chromID C. difficile 100

TCCFA 87
100/96 Alcohol chromID C. difficile 99
TCCFA 96
Shin and Lee, 2014 (72) 530/180 Alcohol chromID C. difficile 55.6 85
CDSA 19.4 75.6

Yang et al., 2014 (73) 289/49 None chromID C. difficile 93.9 98
CCFA 18.4 30.6
Han et al., 2014 (71) 185/36 Heat chromID C. difficile 58.3 100
CDSA 83.3
aTCCA, brain heart infusion agar plus 5% blood, taurocholate, cycloserine, and cefoxitin; CLO, Clostridium
difficile agar (bioMérieux); TCCFA, cycloserine-cefoxitin-fructose-egg yolk agar (CCFA) plus 0.1%
taurocholate; CDSA, C. difficile selective agar (BBL); CCFA, cycloserine-cefoxitin-fructose-egg yolk agar.
plus a simple disk test for pyroglutamyl aminopeptidase, and this may be useful for
laboratories without access to MALDI-TOF MS (77). Furthermore, a subset of C. difficile
strains fails to generate black colonies due to the absence of the ␤-glucosidase gene,
and this appears to be a consistent feature of strains of ribotype 023 (78, 79). In a UK
study, the proportion of isolates failing to generate black or gray colonies within 48 h
was reported to be 1.6% (13). Such isolates may still be detected on chromID C. difficile
due to their characteristic colony shape, but care must be taken to ensure that such
colonies are not overlooked.
It is worth emphasizing that culture of C. difficile alone has little predictive value for
diagnosis of CDI without subsequent demonstration of the isolate’s ability to produce
cytotoxin. This can be directly demonstrated by testing culture supernatants on cell
lines or may be inferred much more rapidly by testing colonies for toxin genes by PCR
(80). Darkoh et al. claimed that they circumvented this problem with the report of a
new chromogenic medium (the Cdifftox plate assay) on which toxigenic C. difficile
isolates formed blue colonies, thus differentiating them from nontoxigenic isolates,
which formed white colonies (81). This was achieved using 5-bromo-4-chloro-3-indolyl-
␤-D-galactopyranoside (X-Gal) for detection of the glycosyltransferase activity of toxins
A and B. Despite its promise, I am not aware of any published evaluation data for this
medium since it was first described in 2011 (81).
Campylobacter spp.
Campylobacter spp. are the most common bacterial cause of gastroenteritis (GI) in
many countries, and infection is primarily due to ingestion of contaminated food. CASA
(Fig. 1d) is a chromogenic medium initially designed for the isolation of Campylobacter
spp. from food, but it has since been evaluated with stool samples from patients with
suspected gastroenteritis. The chromogenic substrate used for detection of Campylo-
bacter spp. is undisclosed by the manufacturer. Le Bars et al. compared CASA with two
nonchromogenic agars (Karmali medium and Campylosel) for the isolation of Campy-
lobacter spp. from 370 diarrheic stool samples (14). Cultures on all three media were
incubated for up to 96 h in a microaerophilic atmosphere at two different temperatures,
37°C and 42°C. The sensitivity of CASA was equivalent to or slightly better than that
shown by either of the other two media, but CASA was reported to be much more
selective, which led to a reduction in the time required for processing colonies for

confirmation of Campylobacter. Dalziel et al. reported the culture of 979 stool samples
on CASA and modified charcoal-cefoperazone-deoxycholate agar (CCDA) with incuba-
tion of cultures at 42°C for 36 to 48 h in a microaerophilic atmosphere (82). The authors
reported sensitivities of 100% and 84% for CASA and CCDA medium, respectively (P ⬍
0.001), and also reported a higher selectivity of CASA. A limitation of CASA is the lack
of a specific chromogenic reaction to indicate the presence of Campylobacter spp., as
other species that grow on the medium also hydrolyze the chromogenic substrate to
produce pink or red colonies, and some may therefore appear quite similar to Campy-
lobacter spp.
Salmonella spp.
Salmonella spp. remain one of the most important causes of foodborne gastroenteritis,
and chromogenic media designed for the specific isolation of Salmonella spp. have been
available for at least 25 years. Rambach agar and SM-ID were the first two examples of such
media (1), and both have been superseded by new generations of media. The principles of

Salmonella detection exploited in these first generations of chromogenic media have been
previously reviewed (50). It has been consistently demonstrated that chromogenic media
do not offer a superior sensitivity to conventional agars such as xylose-lysine-deoxycholate
(XLD) agar and Hektoen enteric agar (83, 84). Furthermore, in contrast to almost all
chromogenic media, such conventional agars offer the opportunity to isolate both Salmo-
nella and Shigella using a single culture medium. No single culture medium or combination
of media can preclude the necessity for enrichment of stool samples, e.g., in selenite broth,
which is essential for detection of low numbers of Salmonella and significantly enhances
detection (84). The sole advantage of chromogenic media for Salmonella is the significantly
higher specificity they afford compared to conventional media. This means that fewer
confirmatory tests than with conventional media are required to investigate colonies of
other species that may resemble Salmonella. This can result in cost savings for laboratories;
e.g., in one study, a saving of EUR 2.7 (approximately US$3) per sample by inclusion of a
chromogenic medium was projected (83).
Most of the chromogenic media for Salmonella that are currently marketed rely on
detection of a C8-esterase enzyme produced by Salmonella that is detected by inclu-
sion of a chromogen linked to caprylic (octanoic) acid. Substrates for ␤-glucosidase
and/or ␤-galactosidase are included so that other coliforms generate a different color.
Antibiotics such as cefsulodin and novobiocin may be included for the inhibition of
Pseudomonas spp. and Proteus spp., respectively. Brilliance Salmonella agar incorporates
a “suicide substrate” or “Inhibigen” that is hydrolyzed by E. coli to release a toxic
product, thus inhibiting its growth (P. Druggan, 21 March 2002, patent application
WO0222785). Only two published studies in the last decade have compared chromo-
genic media for the isolation of Salmonella (83, 84). The chromogenic media included
were chromID Salmonella ELITE, BBL CHROMagar Salmonella, SM-ID2, and Brilliance
Salmonella agar, and these were compared with conventional agars. Neither study
reported any significant difference between any of the media with respect to sensitivity,
but the specificity of chromogenic media was significantly higher than that afforded by
conventional agars.
In summary, the use of a conventional agar (e.g., XLD agar) is appropriate for direct
culture of stool samples, as it accommodates isolation of Shigella spp., and the evidence
suggests that sensitivity is at least equivalent to that of chromogenic media for
detection of Salmonella spp. The use of a chromogenic medium after enrichment in
selenite broth is an attractive option to target Salmonella spp. with high specificity and
consequently reduce the number of colonies requiring investigation. There is no clear
advantage of any particular chromogenic medium for this purpose.
Shigella spp.
Shigella spp. produce few hydrolytic enzymes that provide useful differentiation
from other bacteria, and that has restricted the development of chromogenic media for
their detection. However, the observation that all species of Shigella produce

␤-ribosidase has provided one alternative means of detection (85). HardyCHROM SS is

the only commercially available chromogenic medium allowing isolation of both
Salmonella and Shigella that has been evaluated with clinical samples. Hinde et al.
evaluated this medium with 400 stool samples in comparison with conventional
MacConkey and Hektoen enteric agars (16). The authors reported a superior specificity
for HardyCHROM SS that allowed fewer colonies requiring investigation, and they also
reported a shorter time to detection. However, since only one isolate of Shigella spp.
was recovered during the study, further studies with larger numbers of positive
samples are essential.
Shiga Toxin-Producing Escherichia coli

Shiga toxin-producing E. coli (STEC) bacteria are a cause of foodborne gastroenteri-
tis, hemorrhagic colitis, and hemolytic-uremic syndrome. Among STEC strains, Shiga
toxin production was first associated with E. coli of serotype O157:H7, and it was noted

that such strains, unlike most other E. coli strains, failed to ferment sorbitol. This led to
the design of sorbitol MacConkey agar, which has become widely used by clinical
laboratories. This situation has become much more complicated, and STEC can be
found within many other serotypes that together may account for as much STEC-
associated disease as O157:H7 (86). The fact that many of these additional serotypes
typically ferment sorbitol has severely limited the effectiveness of sorbitol MacConkey
agar for diagnosis of infection with STEC.
Recovery of STEC by culture is challenging due to a low density of organisms in
some stool samples and the lack of consistent biochemical features among STEC
serotypes (87). Despite this, since the first report of Rainbow agar O157 in 1998 (88),
chromogenic media have been commercialized for the detection of STEC. Most of
these media have been designed primarily for isolation of E. coli O157:H7, including
CHROMagar O157, Colorex O157, and Rainbow O157, whereas CHROMagar STEC
allows for detection of a wider range of STEC serotypes (89). Most of these media
are based on similar principles, relying on an inability of E. coli O157 to produce acid
from sorbitol and/or rhamnose and a lack of ␤-glucuronidase activity. A second
chromogenic substrate (e.g., for ␣- or ␤-galactosidase) may be used to highlight the
presence of E. coli O157:H7 among nonreactive background flora (18).
There have been six published evaluations of these media with clinical samples over
the last decade. Grys et al. tested 204 stool samples using PCR for detection of toxin
genes and by direct culture on CHROMagar O157; only four positive samples were
found (all positive by PCR), and three of these were detected by culture (12). Hironven
et al. tested 47 fecal samples from patients with hemorrhagic diarrhea by plating on
CHROMagar STEC and using an immunochromatographic assay for O157 antigen and
Shiga toxin. The chromogenic medium detected STEC in 16 positive samples, compared
with only 14 detected by the immunoassay (90). Wylie et al. compared direct culture on
CHROMagar STEC with a standard cytotoxin assay using 205 fecal samples (86). There
were 14 positive samples, and the sensitivity and specificity of CHROMagar STEC were
reported as 85.7% and 95.8%, respectively. Gouali et al. cultured 329 stool samples onto
CHROMagar STEC and Drigalski agar after preenrichment of the samples in a nonse-
lective broth. Colonies from Drigalski agar were harvested and tested by PCR for toxin
genes (87). From 39 Shiga toxin-positive stool specimens, STEC was recovered as mauve
colonies from 32 samples (sensitivity, 82.1%). Forty-eight isolates of E. coli that were not
found to harbor toxin genes were recovered as mauve colonies on CHROMagar STEC.
McCallum et al. tested 282 fecal samples using PCR for toxin genes and culture on
cefixime-tellurite-sorbitol MacConkey agar (CT-SMAC) and CHROMagar STEC (91). Only
six positive samples were found, of which three were detected using CHROMagar STEC
and only one detected using CT-SMAC, whereas all six were positive using PCR. Finally,
Zeylas et al. tested 536 samples using direct inoculation of CHROMagar STEC and a PCR
test for toxin genes following preenrichment in MacConkey broth (89). Thirteen sam-
ples were found to be positive, and all were detected using PCR. Eleven (84.6%) were

detected by culture on CHROMagar STEC, and a further 68 false-positive colonies were

recovered, giving a low positive predictive value of 13.9%.
The available evidence suggests that chromogenic media for the detection of STEC
do not have sufficient sensitivity or specificity to replace methods that directly detect
toxin or toxin genes in stool samples. Consequently most studies have concluded that
the optimal use of such media is for the isolation of STEC from samples that are
determined positive using more sensitive methods, e.g., PCR (86, 87, 89, 90).
Vibrio spp.
Media for the isolation of pathogenic Vibrio spp., which have been designed
primarily for screening food samples, have been evaluated for use with clinical samples
(15, 92). CHROMagar Vibrio and chromID Vibrio both allow for the isolation and
differentiation of the two most important pathogenic species, Vibrio cholerae and Vibrio
parahaemolyticus, and accommodate isolation of other Vibrio species. The chromogenic
substrates used in these media are undisclosed by the manufacturers. CHROMagar

Vibrio was compared with thiosulfate-citrate-bile salts-sucrose agar (TCBS) for the
isolation of V. parahaemolyticus from 57 patients with suspected gastroenteritis who
had recently ingested seafood. After enrichment in alkaline peptone water, five con-
firmed isolates of V. parahaemolyticus were recovered on CHROMagar Vibrio compared,
with only one isolate on TCBS. There were no false positives on either medium (15).
chromID Vibrio was evaluated against TCBS before and after enrichment in alkaline
peptone water using 28 fecal samples and 66 artificially “spiked” fecal samples. There
was equivalent sensitivity of the two media for isolation of both V. cholerae and V.
parahaemolyticus, but the specificity of chromID Vibrio was reported to be twice that of
TCBS (100% versus 50%) (92). Further studies are required with clinical samples,
including studies in low-prevalence settings.
Yersinia enterocolitica
Y. enterocolitica is a foodborne pathogen and a cause of diarrhea and pseudoap-
pendicitis. The most widely used conventional agar for detection of this pathogen in
stool samples is cefsulodin-irgasan-novobiocin (CIN) agar, which utilizes mannitol
fermentation as a biochemical indicator for Y. enterocolitica. CIN agar is effective, but its
specificity is limited as a number of other species of Enterobacteriaceae are able to grow
on the medium and ferment mannitol, thus also generating magenta colonies (e.g.,
Serratia spp., Providencia spp., Klebsiella oxytoca, and Citrobacter freundii). Weagant
described the development of Yersinia enterocolitica chromogenic medium (YeCM)
(93). This medium sought to improve on the specificity of CIN agar by utilizing
cellobiose as the fermentable carbohydrate for detection of Y. enterocolitica and by
including a chromogenic substrate for ␤-glucosidase, an enzyme that is produced by
most other species of Enterobacteriaceae that are able to grow on CIN agar (93). As well
as enabling differentiation of Y. enterocolitica from other species, this medium had the
additional advantage that only pathogenic types of Y. enterocolitica would be detected
(as nonpathogenic biovars produce ␤-glucosidase). There are no reports of the use of
this medium with human clinical samples, although YeCM was used in a later study that
examined the presence of Y. enterocolitica in 900 tonsil swabs from pigs (94).
Renaud et al. described the use of CHROMagar Yersinia (CAY) (Fig. 1e) for the
isolation of Y. enterocolitica from 1,494 stool samples from hospitalized patients and
used CIN agar as a comparator (17). Although the composition of this medium is
undisclosed, the medium achieves outcomes very similar to those obtained with YeCM,
suggesting the inclusion of a substrate for ␤-glucosidase to increase specificity (95). Six
isolates of pathogenic Y. enterocolitica were successfully isolated using both CAY and
CIN agar, but CAY showed a much higher specificity (99%) than CIN agar (90.4%), with
only 14 false-positive isolates recovered on CAY (P ⬍ 0.001). In contrast, 137 isolates
belonging to other species (predominantly C. freundii and Providencia spp.) grew as
false-positive colonies on CIN agar, and there was no differentiation between the six
pathogenic Y. enterocolitica isolates recovered and six additional nonpathogenic iso-

lates of biovar 1A on CIN agar. Another chromogenic agar, Yersinia Enterocolitica Agar
(YECA), has been described, but its composition is undisclosed and there are no data
available for testing of human samples (96).
CHROMOGENIC MEDIA FOR DETECTION OF ANTIMICROBIAL-RESISTANT

BACTERIA
Prior to 2006, chromogenic media were already available for the specific detec-
tion of methicillin-resistant S. aureus (MRSA) (5). The last decade has seen an
expansion in the range of culture media developed specifically for the detection of
other antimicrobial-resistant bacteria. Between 2007 and 2009, chromogenic media
were first reported for the detection of vancomycin-resistant enterococci (VRE) (8),
Enterobacteriaceae with extended-spectrum ␤-lactamases (7) and carbapenemases
(9), and carbapenem-resistant Acinetobacter spp. (10). Rapid and efficient detection
of these bacteria can allow for prompt decisions regarding the management of

colonized patients in accordance with local infection control policies. Precautions
such as isolation of colonized patients may limit the nosocomial transmission of
antibiotic-resistant bacteria between patients and within the hospital environment.
Methicillin-Resistant Staphylococcus aureus

MRSA strains exhibit resistance to most ␤-lactam antibiotics and frequently show
resistance to other classes, such as macrolides and quinolones. MRSA is a significant
nosocomial pathogen and has been implicated in numerous outbreaks of infection.
Significant efforts have been made to control the spread of MRSA within the hospital
environment, and some authorities advocate universal screening of patients to identify
those who are asymptomatically colonized (97). A great deal of investment has there-
fore focused on optimal diagnostic methods for MRSA, including chromogenic media.
Chromogenic media for MRSA evolved from media designed for detection of all S.
aureus by the simple inclusion of an antimicrobial that inhibits methicillin-susceptible
isolates. The traditional agent of choice for this purpose was oxacillin, and this was
incorporated into CHROMagar S. aureus to create the first chromogenic medium for
detection of MRSA (5). Later studies demonstrated that cefoxitin was a superior option
for selection of MRSA due to induction of methicillin resistance in isolates that showed
heterogeneous expression of resistance (98). A range of chromogenic media has been
commercialized and evaluated with clinical samples, including CHROMagar MRSA (99),
chromID MRSA (100), MRSASelect (101), MRSA-screen (100), Brilliance MRSA (102), and
Spectra MRSA (103). Such media are widely used; for example, in an external quality
assessment exercise in 23 countries in Europe and in Israel, it was reported that 88% of
laboratories utilized a chromogenic medium alone to screen for MRSA (104).
A survey of the literature since 2006 reveals at least 60 publications that have
compared chromogenic media for MRSA with alternative methods using human clinical
samples, and at least 25 of these include a head-to-head comparison of two or more
chromogenic media. The conclusions of these studies are often conflicting, and an
analysis of the data is further confounded by the fact that certain brands of media have
been the subject of incremental improvements to produce newer generations of
these media. When assessing the performance of chromogenic media for MRSA, it
is prudent to analyze studies that are recent and include large numbers of clinical
samples (ideally ⬎1,000 samples).
There is a general consensus that chromogenic media show greater sensitivity than
conventional agars such as mannitol salt agar plus oxacillin. The sensitivity of chromo-
genic agars is increased by incubating plates for 48 h, at the expense of a decrease in
specificity. These observations are confirmed by a meta-analysis of 29 studies reported
between 2004 and 2008 (105). Table 4 summarizes the findings of the five largest
evaluations published since 2010 that compare two or more chromogenic media using
clinical samples (106–110). From this selection of data and from the wider literature, it
is difficult to conclude with any certainty that any particular chromogenic medium is
superior or inferior to its competitors. A number of studies have shown conclusively

TABLE 4 Summary of selected studies evaluating chromogenic media for the isolation of MRSA from patient samples
Sensitivity (%) Specificity (%)
at: at:
Study authors, yr Total no. of
(reference) samples/no. positive Sample type(s) Test medium 24 h 48 h 24 h 48 h
Yang et al., 578/99 Nasal swabs MSA-Fxa 92.9 96 97.1 95.2
2010 (106) MRSASelect 94.9 100 98.5 97.7
MRSA-ID 90.9 99 98.1 97.9
CHROMagar MRSA 91.9 99 99.5 99
Morris et al., 6,035/147 Nasal, groin, axilla, and Brilliance MRSA 2 78.2 99.9
2012 (107) wound swabs chromID MRSA 93.2 99.8
chromID MRSA 2 83.7 99.9
Colorex MRSA 87.1 99.9
Denys et al., 515/73 Nasal swabs BBL CHROMagar MRSA II 87.7 98.6
2013 (108) MRSASelect 89 93.4
Spectra MRSA 83.6 92.1

Veenemans et al., 1,368/102 Nasal, throat, and rectal Direct inoculation
2013 (109) swabs Brilliance MRSA 2 65.7 99.8
MRSA-ID 52 99.2
After broth enrichment
Brilliance MRSA 2 100 99.1
MRSA-ID 98 98.7
Dodémont et al., 1,220/107 Nasal, throat, perineal, and Direct inoculation

2015 (110) skin swabs Brilliance MRSA 2 60.7 72.9 99.7 97.9
chromID MRSA 50.5 71 99.3 96.8
chromID MRSA SMART 66.4 78.5 99 97.8
After broth enrichment
Brilliance MRSA 2 85 98.7
chromID MRSA 87.9 99
chromID MRSA SMART 86 97.8
aMSA-FX, mannitol salt agar plus 5 ␮g/ml cefoxitin.
that the use of broth enrichment prior to inoculation onto chromogenic agar can
significantly increase the sensitivity of MRSA detection, and this is exemplified by two
of the studies in Table 4 (109, 110).
Vancomycin-Resistant Enterococci
chromID VRE was the first chromogenic medium reported for the isolation of
enterococci with acquired resistance to glycopeptides (e.g., vancomycin and teicopla-
nin) (8). In six studies with stool samples or rectal swabs, chromID VRE was compared
with bile-esculin agars (with or without azide) supplemented with vancomycin (8,
111–115). Two of these studies reported a superior sensitivity of chromID VRE (8, 115),
and the others reported equivalent sensitivity (111–114). A consistent advantage of
chromID VRE was its ability to differentiate between Enterococcus faecalis and Entero-
coccus faecium. This is achieved by incorporating chromogenic substrates for detection
of ␣-glucosidase and ␤-galactosidase (18). chromID VRE also showed a higher speci-
ficity, leading to a reduction in the number of confirmatory tests that were required for
suspect colonies. Five other chromogenic media, AES VRE agar (115), Brilliance VRE (Fig.
2a) (116), CHROMagar VRE (117, 118), Spectra VRE (119–121), and VRESelect (122), have
since been evaluated against bile-esculin agars (with or without azide) supplemented
with vancomycin, in studies with stool samples or rectal swabs. In each of the eight
cited studies, the chromogenic media offered higher sensitivity and specificity. All
except AES VRE agar provided differentiation of E. faecalis from E. faecium.
Relatively few studies have included head-to-head comparisons of chromogenic
agars for the isolation of VRE from clinical samples (115, 123–125). CHROMagar VRE and
chromID VRE were evaluated with 259 stool samples after an overnight enrichment
step and a 48-h incubation period (123). The authors reported an identical sensitivity of
the two media (98.2%) and an equivalent specificity. Suwantarat et al. performed a


FIG 2 Examples of chromogenic media for detection of antimicrobial-resistant pathogens that have been
first reported in the last decade. (a) Purple colonies of Enterococcus faecium (vanB) and blue-green
colonies of Enterococcus faecalis (vanA) on Brilliance VRE agar. (b) ESBL-producing colonies of Klebsiella
pneumoniae with SHV-36 and CTX-M-9 enzymes (green colonies) and Escherichia coli with CTX-M-9
enzyme (pink/blue colonies) on Brilliance ESBL agar. (c) K. pneumoniae (green colonies) and E. coli (red
colonies), both with OXA-48 carbapenemase, isolated on chromID OXA 48. (d) Carbapenemase-
producing K. pneumoniae with OXA-48 enzyme (blue colonies) and E. coli with NDM-1 enzyme (pink
colonies) isolated on Colorex mSuperCarba medium.
large study that compared five chromogenic agars with bile-esculin-azide-vancomycin

agar (BEAV) using 400 stool samples, of which 99 contained VRE (124). The chromo-
genic media comprised InTray Colorex VRE, chromID VRE, VRESelect, HardyCHROM VRE,
and Spectra VRE. The authors reported a significantly higher sensitivity (89.9 to 93.9%)
of all chromogenic agars than of BEAV (84.8%) and also an earlier time to detection.
chromID VRE showed the highest sensitivity of the five chromogenic agars, but this was
not statistically significant. Four of the chromogenic agars showed identical specificity
(99.7%), whereas the specificity of InTray Colorex VRE was lower (98.3%). Differences
were noted among the chromogenic media with respect to time to detection, the need
for supplementary testing, and ease of color distinction among colonies, as well as
non-VRE breakthrough growth. Finally, Gouliouris et al. compared chromID VRE and
Brilliance VRE for the isolation of VRE from 295 stool samples from nursing home
residents (125). They reported an equivalent sensitivity of the two chromogenic media
and noted that the sensitivity of both media was significantly improved by incubation
for 48 h and inclusion of a preenrichment step. The selectivity of Brilliance VRE was
higher than that of chromID VRE.
In conclusion, chromogenic media for detection of VRE offer a sensitivity that is
frequently reported as better than that of conventional media such as BEAV. Unlike
BEAV, most chromogenic media allow differentiation and presumptive identification of
the two dominant species of enterococci, and most reports note that less time is
required for processing of colonies due to the higher selectivity/specificity of these
media. There is no clear difference in sensitivity among most of the chromogenic agars
reported here.

TABLE 5 Summary of studies evaluating chromogenic media for the isolation of Enterobacteriaceae with extended-spectrum-␤-lactamases
from clinical samples
Positive
Sensitivity (%) predictive value
at: (%) at:
Study authors, yr Total no. of
(reference) samples/no. positive Sample type(s) (n) Test medium 18–24 h 48 h 18–24 h 48 h
Réglier-Poupet et al., 765/33 Rectal swab (468), urine chromID ESBL 88 94 39 28
2008 (128) (255), respiratory (42) BLSE 85 85 15 11
Huang et al., 528/59 Fecal (344), respiratory (134), chromID ESBL 94.9 48.7
2010 (129) miscellaneous (50) Brilliance ESBL 94.9 46.7
MAC with ceftazidime disk 74.6 64.7
Paniagua et al., 500/41 Stool (500) chromID ESBL 100 63

2010 (130) MAC ⫹ 1 ␮g/ml CAZ plus 87.8 43.4
MAC ⫹ 1 ␮g/ml CTX

Saito et al., 256/17 Stool (186), urine (48), other chromID ESBL 88.2 46.9
2010 (131) (22) CHROMagar ESBL 100 51.5
Willems et al., 139/16 Perineal and nasal swabs chromID ESBL 81.2 87.5 35.1 33.3
2013 (132) (139) Brilliance ESBL 87.5 87.5 38.9 33.3
BLSE 87.5 87.5 22.2 20.3
Grohs et al., 2,337/354 Rectal swab (2,337) chromID ESBL 97.5 39.1
2013 (133) Brilliance ESBL 98.6 29.5
CHROMagar ESBL 98.3 38.7
Drigalski ⫹ CAZ 97.2 32.5
Drigalski ⫹ CTX 95.5 29.5
Blane et al., 298/116 Stool (298) chromID ESBL 63 75

2016 (134) Brilliance ESBL 59 68
aBLSE,A commercially available biplate comprising Drigalski agar (with 1.5 ␮g/ml cefotaxime) and MacConkey agar (with 2 ␮g/ml ceftazidime); MAC, MacConkey agar;
CAZ, ceftazidime; CTX, cefotaxime; Drigalski ⫹ CAZ, BD Drigalski lactose agar with ceftazidime; Drigalski ⫹ CTX, Drigalski agar plus 2 ␮g/ml cefotaxime.
Extended-Spectrum-␤-Lactamase-Producing Enterobacteriaceae
Extended-spectrum ␤-lactamases (ESBLs) have become globally disseminated within
species of Enterobacteriaceae. These enzymes hydrolyze third-generation cephalosporins,
typically conferring resistance to these agents, but are inhibited by clavulanic acid. Genes
encoding ESBLs are frequently found on transmissible plasmids that often harbor other
resistance determinants (e.g., encoding resistance to aminoglycosides). Prompt and accu-
rate detection of ESBL producers can assist in guiding optimal antimicrobial therapy (126)
and limiting nosocomial transmission (127). The first published report of a chromogenic
medium for detection of ESBL producers described the evaluation of ESBL-Bx (a prototype
of chromID ESBL). The authors compared this new medium with MacConkey agar supple-
mented with 2 ␮g/ml ceftazidime for the isolation of ESBL producers from 644 clinical
samples (7). They reported a sensitivity of 97.7% for the chromogenic medium, compared
with 84.1% for MacConkey agar plus ceftazidime. Since this first report, a range of chro-
mogenic media has been made commercially available, and reports of their performance
are summarized in Table 5 (128–134). The principles of these media have much in
common. The media typically employ a combination of chromogenic substrates to
detect ␤-galactosidase (or ␤-glucuronidase) production by E. coli and ␤-glucosidase
production by the KES group. This allows detection and differentiation of the main
species or groups associated with ESBL production. A cephalosporin is incorporated
to inhibit susceptible strains, and other inhibitors may be included to inhibit the
growth of Gram-positive bacteria and yeasts. Enterobacteriaceae with AmpC
␤-lactamase are frequently isolated on all of these media, and to a large degree this
accounts for the relatively low positive predictive values shown in Table 5 (133).
Five of the studies in Table 5 include a direct comparison of two or more chromo-
genic media, and no significant difference in sensitivity was reported after 24 h of

incubation in any of these studies (129, 131–134). The yield of ESBL producers may be
increased significantly (particularly on chromID ESBL) by extending incubation up to 48
h (128, 134), and a preenrichment step may also significantly increase sensitivity (134).
However, both of these strategies contribute to an even lower specificity (132, 134). In
three of the studies in Table 5 the positive predictive value of chromogenic media was
reported to be significantly higher than that of nonchromogenic comparators (128, 130,
132).
Carbapenemase-Producing Enterobacteriaceae
Perhaps the most significant development in clinical bacteriology over the last
decade has been the global proliferation of Enterobacteriaceae with acquired carbap-
enemase enzymes (carbapenemase-producing Enterobacteriaceae [CPE]) (135). Avail-
able data suggest that the vast majority of carbapenemases in Enterobacteriaceae
belong to one of the five major families: the IMP, NDM, and VIM metalloenzymes and
the Klebsiella pneumoniae carbapenemase (KPC) and OXA-48-like enzymes (136). CPE

are frequently resistant to virtually all ␤-lactam antibiotics (including carbapenems),
and carbapenemase genes are transmissible via plasmids. Concomitant resistance to
several other antimicrobial classes is common in CPE, dramatically reducing treatment
options for infected patients. CPE are frequently implicated in nosocomial outbreaks,
and fecal carriage of CPE is an important reservoir for subsequent transmission (137). In
early 2009, the Centers for Disease Control and Prevention (CDC) and the Healthcare
Infection Control Practices Advisory Committee recommended measures for active
surveillance of Enterobacteriaceae with resistance to carbapenems in all acute-care
facilities in the United States (138). A practical screening method was also recom-
mended by the CDC, which involved inoculation of rectal swabs into 5 ml of Trypticase
soy broth (TSB) along with a 10-␮g ertapenem or meropenem disk (139). After
incubation, the broth is subcultured to MacConkey agar, and lactose-fermenting col-
onies are investigated for carbapenemase production or carbapenem resistance. De-
tection of CPE is not straightforward, as not all carbapenemases confer clinical resis-
tance to carbapenems and Enterobacteriaceae without carbapenemases may exhibit
resistance to carbapenems via other mechanisms (140).
In 2008, Samra et al. evaluated CHROMagar KPC, the first chromogenic culture
medium for detection of CPE (9). The medium was an adaptation of CHROMagar
Orientation with additional selective agents for inhibition of Gram-positive bacteria and
carbapenem-susceptible Gram-negative bacteria. CHROMagar KPC is also available as
prepoured plates and is marketed as Colorex KPC (141). The authors compared culture
on CHROMagar KPC with culture on MacConkey agar (plus carbapenem disks) and a
PCR method for detection of blaKPC genes. The sensitivity and specificity relative to PCR
were 100% and 98.4%, respectively, for CHROMagar KPC and 92.7% and 95.9%,
respectively, for MacConkey agar with carbapenem disks. Since this first report, a
number of other chromogenic media have been made commercially available for
detection of CPE. As with media for the detection of ESBL producers, the principles of
these media have much in common, and they allow for the differentiation of the most
relevant Enterobacteriaceae (i.e., E. coli and the KES group) as well as incorporating
selective agents to inhibit Gram-positive bacteria and yeasts. The choice (and concen-
tration) of antimicrobial(s) selected for inhibition of carbapenem-susceptible Entero-
bacteriaceae is the most critical factor that influences sensitivity and specificity, and the
inclusion of antimicrobials other than carbapenems may have advantages over the
inclusion of a carbapenem (S. Ghirardi, J. D. Perry, and G. Zambardi, 4 October 2012,
international patent application WO2012131217; L. Devigne, S. Ghirardi, and B. Zam-
bardi, 30 January 2014, international patent application WO2014016534). However, the
ingredients of commercially available chromogenic media are typically undisclosed.
There have been a number of published studies that have examined the limit of
detection of chromogenic agars for various types of CPE using challenge experiments
with pure cultures (142–151). While such studies are useful, they cannot replicate the
challenges that may be encountered when seeking to recover CPE from patient

TABLE 6 Summary of studies evaluating chromogenic media for the isolation of carbapenemase-producing Enterobacteriaceae from
patient samples
Study authors, yr Total no. of Sensitivity Specificity Study Dominant
(reference) samples/no. positive Test medium (%) (%) location enzyme(s)
Samra et al., 2008 (9)a 122/41 CHROMagar KPC 100 98.4 Israel KPC
MacConkey agar ⫹ carbapenem disks 92.7 95.9
Adler et al., 2011 (152) 139/33 CHROMagar KPC 84.9 88.7 Israel KPC
MacConkey agar ⫹ imipenem (1 ␮g/ml) 84.9 94.3
Panagea et al., 2011 126/46 CHROMagar KPC 97.8 Greece KPC, VIM
(153) MacConkey agar ⫹ imipenem (1 ␮g/ml) 78.3
Vrioni et al., 2012 (154) 200/73 TSBb ⫹ ertapenem (2 ␮g/ml) 89.1 86.4 Greece KPC, VIM
chromID ESBL 92.4 93.3
chromID ESBL (plus enrichment) 92.4 84.7

chromID CARBA 92.4 96.9
MacConkey agar ⫹ meropenem (1 ␮g/ml) 89.1 85.2
Vasoo et al., 2014 (155) 150/47 CHROMagar KPC 76.6 75.7 USA KPC
Remel Spectra CRE 97.8 86.4
MacConkey agar ⫹ ertapenem disks 83 73.8
Papadimitriou-Olivgeris 177/86 chromID CARBA 96.5 91.2 Greece KPC

et al., 2014 (156) MacConkey agar ⫹ imipenem (1 ␮g/ml) 89.5 31.9
TSB ⫹ ertapenem (2 ␮g/ml) 98.8 80.2
Zarakolu et al., 2015 302/33 chromID CARBA 57.6 98.9 Turkey OXA-48
(157) chromID OXA-48 75.8 99.3
TSB ⫹ ertapenem (2 ␮g/ml) 57.6 95.2
Davies et al., 2016 236/33 Brilliance CRE 97 87.9 UK NDM

(158) chromID CARBA 97 91.5
Colorex mSuperCarba 90.9 92.4
Papadimitriou-Olivgeris 912/329 Brilliance CRE 96.8 90.9 Greece KPC

et al., 2016 (159) CHROMagar KPC 99.2 78.2
MacConkey agar ⫹ imipenem disk 67.2 98.1
MacConkey agar ⫹ ertapenem disk 81.8 97.9
aThe sensitivity and specificity of both media were calculated relative to results obtained by PCR.
bTSB, Trypticase soy broth.
samples. Consequently, the values for sensitivity and specificity reported in evaluations
with clinical samples are usually lower than those achieved with pure cultures (140).
Table 6 summarizes the findings of the largest studies that have been performed with
fecal samples (stool samples or rectal swabs) from patients (9, 152–159). In three early
studies with CHROMagar KPC (9, 152, 153), sensitivity was equivalent to or higher than
that of in-house preparations of MacConkey agar incorporating imipenem (or Mac-
Conkey agar with carbapenem disks). Later studies revealed that isolates producing
NDM-1 carbapenemase may be inhibited by CHROMagar/Colorex KPC if the isolates
have a relatively low MIC to meropenem (ⱕ2 ␮g/ml) (141, 143). This emphasizes the
importance of performing studies in different geographical areas where different
carbapenemase enzymes may predominate. In a further example, chromID CARBA was
proven to be effective in comparative studies of media in Greece (154, 156) and
Pakistan (141, 160, 161) but had limited efficacy in Turkey (157) and Belgium (162),
where OXA-48 is the dominant carbapenemase. To address this, the manufacturer
offers a biplate (chromID CARBA SMART) that combines two media in a single petri dish:
chromID CARBA and chromID OXA-48.
Brilliance CRE agar is marketed for the isolation of carbapenem-resistant Enterobac-
teriaceae (CRE) rather than CPE. This medium was evaluated in two simultaneous
studies in two centers in Pakistan and showed a lower sensitivity and specificity than

chromID CARBA for detection of CPE with NDM-1 carbapenemase (160, 161). The
authors speculated that the stability of Brilliance CRE may have been compromised
during transport of the culture media from the UK to Pakistan. However, in challenge
experiments with pure cultures, a relatively low specificity of 71% was recorded for this
medium due to the growth of AmpC- and/or ESBL-producing isolates (146). In another
study with Brilliance CRE with patient samples (n ⫽ 77), the specificity of Brilliance CRE
was lower than that of SuperCarba (86.6% versus 98.5%, respectively) (149). However,
the sensitivity of Brilliance CRE was significantly better than that of chromID CARBA for
detection of OXA-48 as shown by a study in Belgium (162). The sensitivity of both
media was improved by preenrichment of rectal swabs in MacConkey broth (although
this was not statistically significant), whereas prolonged incubation of media for 48 h
showed no advantage.
SuperCarba medium is a nonchromogenic medium for isolation of CPE that incor-
porates a low concentration of ertapenem (0.5 ␮g/ml) in addition to cloxacillin in a

zinc-supplemented Drigalski-lactose agar (163). Studies with bacterial isolates suggest
a high sensitivity for detection of all types of CPE, but stability of the medium is limited
to 1 week, and larger studies with patient samples are required. A chromogenic
adaptation of this medium, CHROMagar mSuperCarba (also marketed as Colorex
mSuperCarba) (Fig. 2d), has recently been made commercially available and shows a
comparable sensitivity when tested with pure isolates of CPE (164). The medium, once
prepared, has an improved shelf life of 1 month. Davies et al. (158) recently reported an
evaluation of Colorex mSuperCarba with rectal swabs and reported a sensitivity equiv-
alent to that of chromID CARBA and Brilliance CRE for recovery of CPE that mostly
produced NDM carbapenemase (Table 6). García-Fernández et al. cultured 211 rectal
swabs from distinct patients onto CHROMagar mSuperCarba and compared its perfor-
mance with culture on chromID CARBA, chromID OXA-48, and chromID ESBL (165). CPE
were detected in 61 samples (with OXA-48 reported as the dominant enzyme; n ⫽ 54),
and the authors reported 100% sensitivity and specificity for CHROMagar mSuperCarba.
The sensitivities of the comparator media were not reported.
In the three evaluations in Table 6 that included the CDC broth method, the
sensitivity of chromogenic media was equivalent (154, 156) or better (157). Disadvan-
tages of the CDC broth method include a longer time to detection (an extra day is
required), lack of information regarding likely species identification, and the fact that
CPE may not be detected if they fail to ferment lactose on MacConkey agar. A further
disadvantage is that the CDC broth method is significantly more labor-intensive than
use of a chromogenic medium, as shown by Mathers et al. (166).
It can be concluded that chromogenic media for CPE have clear advantages over the
CDC broth method or the use of MacConkey agars supplemented with a carbapenem
or used in conjunction with carbapenem disks. However, it is especially difficult to
establish which, if any, chromogenic medium is optimal for detection of CPE due to the
different types of carbapenemase that may be encountered and the dominance of
particular types in certain geographical regions. In January 2016, Viau et al. published
an extensive evaluation of all methods for the detection of CPE (140). Following a
detailed statistical meta-analysis of published studies, the authors concluded that
chromID CARBA and the nonchromogenic SuperCarba medium have excellent sensi-
tivities for class A ␤-lactamases (e.g., KPC) that rival that of real-time PCR and that the
CDC broth method was generally inferior to chromogenic media. For other media, and
other carbapenemase types, there was insufficient evidence to draw firm conclusions.
Carbapenem-Resistant Acinetobacter spp.

Species of Acinetobacter, and especially Acinetobacter baumannii, are important
nosocomial pathogens that have been responsible for outbreaks of infection among
hospitalized patients. Strains that are resistant to carbapenem antibiotics are particu-
larly problematic, as infections caused by such strains can be very difficult to treat (167).
In 2009 CHROMagar Acinetobacter, the first chromogenic medium for detection of
Acinetobacter spp., was described, and this medium can be used to detect all Acineto-

bacter spp. or it can be further supplemented to detect only isolates with resistance to
carbapenems (10). The chromogenic substrate used for detection of Acinetobacter spp.
is undisclosed by the manufacturer. The authors evaluated the supplemented version
of CHROMagar Acinetobacter for detection of carbapenem-resistant strains from stool
samples and perineal swabs of 70 patients and compared its performance to PCR
following enrichment culture. The sensitivity and specificity of the chromogenic me-
dium compared with the PCR assay were reported as 91.7% and 89.6%, respectively
(10).
There have been few subsequent published evaluations of CHROMagar Acinetobac-
ter with clinical samples for the isolation of carbapenem-resistant isolates. Song et al.
used an updated version of the medium (Fig. 1f) for isolation of carbapenem-resistant
Acinetobacter from 406 paired nasal and rectal swabs (168). They confirmed the high
specificity of the medium for carbapenem-resistant Acinetobacter, but no comparator was
used for assessment of sensitivity. In a study using spiked stool samples, CHROMagar

Acinetobacter had a reported sensitivity of 86.5% and a specificity of 75% for detection of
carbapenemase-producing A. baumannii (169).
One question that deserves further attention is whether a single chromogenic
medium could be used by clinical laboratories to perform screening for both
carbapenemase-producing Enterobacteriaceae and carbapenem-resistant Acinetobacter
(170, 171). For example, Higgins et al. reported that a high proportion of carbapenem-
resistant Acinetobacter strains with a diverse range of carbapenemase enzymes grow on
chromID CARBA and that the medium effectively inhibits the growth of carbapenem-
susceptible isolates (170). Zarakolu et al. evaluated CHROMagar Acinetobacter and chromID
CARBA for the isolation of carbapenem-resistant Acinetobacter with 203 stool samples from
patients at a hospital in Turkey (171). There was no significant difference between the two
media, with sensitivities of 66% and 77%, respectively (P ⫽ 0.48) and comparable values for
positive predictive value (62% and 68%). However, all isolates belonged to a single species
(A. baumannii), and all produced OXA-23 carbapenemase. Further studies are therefore
warranted for evaluation of CHROMagar Acinetobacter in different geographical areas, and
it would be useful if further studies can establish whether separate media are necessary for
screening for CPE and carbapenem-resistant Acinetobacter.
IMPACT OF LABORATORY AUTOMATION ON THE USE OF CHROMOGENIC MEDIA

Another major development of the last decade has been the introduction of
MALDI-TOF MS into many clinical laboratories for rapid and accurate identification
of species (172). In many ways this has proven to be a powerful adjunct to the use of
chromogenic media (and culture methods in general), as culture is a prerequisite for the
majority of MALDI-TOF MS applications in diagnostic microbiology. None of the chro-
mogenic media described in this review allows for absolute specificity in terms of
pathogen detection, and colonies therefore require further identification. The main
benefits of MALDI-TOF MS have been to reduce the time required to less than an hour
for confirmation of pathogens isolated by culture and to reduce the cost of identifica-
tion. Also, as multiple colonies can be quickly screened to provide species-level
identification, the availability of MALDI-TOF MS can compensate, to some extent, for a
lack of specificity of some culture media. This may tempt some to choose less expensive
conventional media rather than chromogenic media, but the availability of MALDI-TOF
MS cannot compensate for any deficit in sensitivity; i.e., it can be applied only to
colonies that have actually been detected in the first place. As well as providing species
identification, the confirmation of ESBL or carbapenemase enzymes using MALDI-TOF
MS is also possible (173, 174). A number of studies have confirmed the value and
compatibility of MALDI-TOF MS when used in conjunction with many of the chromo-
genic media described in this review (see, e.g., references 46, 74, 158, and 175).
Innovative applications of other forms of mass spectrometry that demonstrate the
feasibility of detecting a broad variety of resistance enzymes directly from limited
biomaterials have also been published (176).
Other developments in laboratory automation also strengthen the continued role of

culture media, and chromogenic media in particular, in the clinical laboratory. These
include automated methods for inoculation of culture plates (177) and methods for
automated digital analysis that enable detection and enumeration of colored colonies
(178, 179). Digital imaging software is capable of distinguishing differences in pixel
color, and chromogenic media are particularly suited to digital automation, as color
thresholds can be created to detect target growth of specific pathogens (179). Faron et
al. exploited this technology to screen for MRSA in a multicenter study using the
WASPLab chromogenic detection module (179). Three different chromogenic media,
MRSASelect, CHROMagar MRSA, and chromID MRSA, were used, and cultures from
57,690 screening swabs were interpreted by manual reading of digital images and by
automated digital analysis. Automated analysis demonstrated 100% sensitivity for all
three chromogenic media and 90.7% specificity, indicating that automated analysis was
highly reliable for reporting negative samples (which constituted 88.6% of total sam-
ples in this study). Technologist support was required for examination of cultures

declared “positive” by automated analysis to rule out false positives. A total of 5,057
false-positive results were reported by the automated digital analysis (8.8% of total
samples). Of interest, automated digital analysis detected colored colonies in 153
positive cultures that were “missed” by the first manual reading but declared positive
when discrepant results were reviewed manually (179).
Faron et al. reported on the use of the same technology for detection of VRE in
104,730 rectal swabs cultured onto Colorex VRE and Oxoid VRE (178). The sensitivity
and specificity of automated digital analysis were 100% and 89.5% compared with
manual interpretation of digital images, and the automated method correctly identified
all negative cultures, which comprised 84% (87,973) of the specimens in this study.
There were discordant results for 10,348 specimens, and these were reexamined
manually. This reexamination revealed 499 positive cultures that were missed by the
first manual reading. Most of the other discordant results were falsely identified as
positive by the automated system and were attributed to residual specimen matrix,
yeast growth, or colonies with borderline color development (178). The authors calcu-
lated that the application of automated specimen processing and reading of cultures
allowed a saving of approximately $5 per negative sample due to the reduction in labor
time. The issue of automatic digital plate reading for surveillance cultures is the subject
of a recent commentary (180).
CULTURE USING CHROMOGENIC MEDIA VERSUS MOLECULAR DIAGNOSTIC

METHODS
A wide range of molecular diagnostic assays, including PCR, microarrays, and
whole-genome sequencing, is becoming available to diagnostic clinical laboratories.
Clinical microbiologists are increasingly required to assess the relative merits and
clinical impact of such methods against those of culture, using either conventional or
chromogenic media. The primary consideration in such decision-making is the relative
sensitivities of competing tests, and a number of investigators have sought to elucidate
the relative sensitivity of molecular methods versus culture using chromogenic media
for direct testing of clinical samples. Evidence suggests that PCR and culture using
chromogenic media have equivalent sensitivity when used as screening tests for C.
difficile (181, 182) and vancomycin-resistant enterococci (183–186). However, culture for
C. difficile requires subsequent confirmation of toxin genes, whereas PCR has the
advantage of specifically detecting toxigenic C. difficile. For detection of S. agalactiae,
there is evidence that PCR methods are more sensitive than direct culture on chromo-
genic media (38, 187); however, there is no significant difference when PCR is compared
with culture on chromogenic media after broth enrichment (38, 188–190).
Infectious causes of gastroenteritis (GI) may include several bacterial pathogens and
a range of viruses and parasites. Laboratory diagnosis by conventional methods is
therefore cumbersome and requires a combination of methods that commonly in-
cludes culture (including enrichment broth[s]), microscopy, and immunoassays. For
some GI pathogens, e.g., STEC, there is a clear advantage of molecular assays compared

to any form of culture, as discussed above. De Boer et al. employed a multiplex PCR
assay to target four bacterial gastrointestinal pathogens (Salmonella spp. Campylobac-
ter jejuni, STEC, and Shigella/enteroinvasive E. coli) in 13,974 stool samples. They
reported that the molecular assay markedly improved rates of detection of all patho-
gens apart from Salmonella spp. compared to culture on conventional media (191). The
use of commercial platforms that employ multiplexed molecular assays and can
accommodate a large number of targets is an increasingly attractive option to simplify
laboratory workflow. One example of such a platform is the FilmArray GI panel, which
consists of automated nucleic acid extraction, reverse transcription, amplification, and
analysis and can detect 22 pathogens, with results available within 1 h. In a multicenter
study, Buss et al. examined the performance of this panel for detection of a range of GI
pathogens in 1,556 stool samples (192). Using conventional culture (with nonchromo-
genic media) as a comparator, the molecular assay showed high sensitivity (94.5 to
100%) for detection of Salmonella, Yersinia, STEC, Campylobacter, C. difficile, and Shi-

gella, as well as other pathogens. There was also evidence that certain pathogens (e.g.,
Campylobacter upsaliensis and Plesiomonas shigelloides) were poorly detected by cul-
ture (192). In conclusion, there is good evidence that molecular platforms offer high
sensitivity and versatility for the convenient detection of a range of GI pathogens (193),
and there is evidence of accelerated uptake of these tests, particularly in the United
States (194). Although most comparative studies have not included chromogenic
media, any advantage of chromogenic media over conventional media for detection of
GI pathogens is unlikely to be a major consideration in deciding whether to use culture
or a molecular platform.
A considerable amount of work has focused on the potential advantages of PCR for
the rapid detection of patients colonized with MRSA. In 2011, Luteijn et al. published
a systematic review and meta-analysis of 29 studies that compared PCR with culture on
chromogenic media for detection of MRSA in screening swabs (105). There was no
significant difference between the two methods if results of chromogenic culture were
considered after incubation for 48 h. Subsequent studies have shown conflicting
results, with chromogenic media found to be equivalent (195–197), inferior (198), or
superior (199–201) to a variety of PCR methods. The obvious potential advantage of
PCR over culture is the significantly reduced turnaround time required for detection of
MRSA. For example, Roisin et al. reported that use of PCR led to a reduction in the
median turnaround time from admission to notification of positive results from 88 h to
11 h and reduced the median time between admission and isolation of newly detected
MRSA carriers from 96 h to 25 h (202). Despite this, a reduction in nosocomial
transmission of MRSA was not demonstrated. Reductions in turnaround time and the
number of days required for patient isolation were also confirmed by a systematic
review by Polisena et al. (203). In a major study, Derde et al. examined the impact of
screening using PCR versus screening using chromogenic media to examine the
acquisition and transmission of MRSA (and other resistant bacteria) on 13 intensive care
units in eight countries. The authors could not find any positive impact of PCR on
acquisition rates (204).
The accumulated evidence suggests that chromogenic media have a sensitivity that
is comparable to that of PCR for detection of MRSA if cultures are incubated for 48 h
or enrichment culture is included. PCR offers the advantage of a significantly faster
turnaround time and may facilitate prompt isolation of colonized patients and/or a
reduced number of days of isolation. Despite this, there is no evidence of a reduced rate
of MRSA acquisition. The ongoing debate surrounding the need for, and extent of,
screening of patients for MRSA is beyond the scope of this review (97).
There are a very limited number of studies that have compared PCR with culture on
chromogenic media for detection of CPE directly from clinical samples. As noted
previously, it is likely that the two methods have comparable sensitivity for detection
of strains harboring KPC carbapenemase (9, 140). However, there is at least circum-
stantial evidence that isolates with other carbapenemases (and perhaps lower levels of
resistance to carbapenems) may be more readily detected by molecular methods such

as PCR. In a study in Belgium, Huang et al. applied the Check-Direct CPE PCR assay for
the testing of 394 rectal swabs to detect CPE carriage and also cultured the swabs on
two chromogenic media (chromID CARBA/chromID OXA-48) (205). They reported that
CPE was detected by PCR in 38 samples but that only 17 samples yielded CPE by
culture. OXA-48 was the dominant carbapenemase enzyme. Of the 21 samples that
were positive by PCR but negative on culture, five of the samples were from patients
with previous carriage of CPE, and a further six samples (all with OXA-48 detected by
PCR) were from patients located in a center where a longstanding outbreak of OXA-
48-producing Klebsiella pneumoniae was occurring. It is possible that this study dem-
onstrates a superior sensitivity of PCR for detection of CPE, although the possibility that
carbapenemase genes were detected in species other than Enterobacteriaceae that may
not be targeted by chromogenic media cannot be excluded (206, 207). Otter et al.
performed screening of 4,006 patients for CPE using PCR (Check-Direct CPE assay) and
culture on a chromogenic medium (chromID CARBA SMART) at a hospital in London, UK

(208). Culture was also performed on all samples using a conventional agar (MacConkey
agar plus a 10-␮g ertapenem disk), and samples that were positive using PCR but
negative by culture were screened for CPE using two different enrichment broths. Using
culture, only six CPE were recovered from five patients (0.1%). Five CPE were detected
using the chromogenic medium, compared with only one using the conventional agar,
whereas the PCR assay failed to detect two CPE with OXA-48 carbapenemase. Samples
from 76 patients were culture negative but were positive using the Check-Direct CPE
assay. No CPE could be recovered from these 76 samples using two types of enrichment
culture, and only 2/76 samples generated a positive PCR test when the same samples
were retested 1 to 2 months later. After assessing evidence from this and other studies,
the authors recommended that a lower threshold cycle (CT) cutoff value of ⬍35 should
be introduced in order to reduce the number of false-positive PCR results. They also
concluded that screening using PCR may not be cost-effective in a setting with such a
low prevalence (208). There is a pressing need for further trials of PCR versus culture on
chromogenic media for detection of CPE.
For other chromogenic applications covered by this review, the lack of published
comparisons with molecular methods using clinical samples precludes any meaningful
analysis.
Other than assessing the test performance, the choice of whether to use a chro-
mogenic culture method or a molecular method will be determined by additional
factors, which most notably include the cost per test (and the cost of associated
instrumentation) and the likely impact of turnaround time on patient management.
PCR tests are generally significantly more expensive than cultures using chromogenic
media, but PCR results are normally available within a few hours, whereas culture
results will be available only after 18 to 48 h. In reality, this is no longer a binary choice,
and an increasing trend in clinical microbiology over the last decade has been the need
to develop diagnostic algorithms that combine two or more complementary tests. This
is well recognized in the diagnosis of C. difficile infection, where a diagnostic algorithm
might conceivably include an enzyme immunoassay(s), chromogenic culture, MALDI-
TOF MS, and PCR for toxin genes (209, 210). Following culture of presumptive colonies
of CPE (or VRE) on chromogenic media, a PCR test is desirable for confirmation of the
presence of a resistance gene and identification of the type of gene allele present.
Conversely, if resistance genes are detected in a rectal swab using PCR, culture is then
necessary in order to identify the species harboring the gene and to perform subse-
quent analyses (e.g., antimicrobial susceptibility testing).
Despite the increasing use of culture-independent tests for detection of GI patho-
gens, culture remains important for isolation of bacterial pathogens that have been
detected by such methods. This enables susceptibility tests to be performed and is
essential for monitoring outbreaks of infection and, where possible, linking such
outbreaks to their primary source, e.g., contaminated food. This issue has come into
sharper focus in the last year, with the CDC characterizing culture-independent tests as
“a serious and current threat to public health surveillance, particularly for STEC and

Salmonella” (194). In the long term, metagenomic approaches may allow reference
laboratories to type enteropathogens directly from clinical samples, but until such tests
are widely available, it is essential that culture methods are utilized for recovery of
pathogens to allow for typing of isolates and investigation of outbreaks. For such
culture methods, the relative attributes of chromogenic versus conventional agars
should be carefully considered.
CONCLUSIONS
The last decade has seen a rapid expansion in the range of chromogenic culture
media available to clinical laboratories. In most cases, a clear advantage over conven-
tional culture media can be demonstrated. This not only is due to the inclusion of
chromogenic substrates that allow detection of pathogens with high specificity but
also is due to the inclusion of optimal combinations of selective agents that prevent
interference from nontarget microorganisms. In some cases, media that have been

designed primarily for the isolation of pathogens (e.g., Campylobacter spp., Y. entero-
colitica, and Vibrio spp.) from food have found application in clinical diagnostics. This
trend continues with a recent report of Listeria chromogenic agar applied to the
diagnosis of neonatal bacteremia (211). New culture media continue to be developed
as research tools, as well as for routine diagnostics, such as the recent report of a
chromogenic medium for isolation of antimicrobial-resistant strains of Bacteroides
fragilis (212). It is also likely that bacteria with new forms of antimicrobial resistance
will be targeted by new chromogenic media. This is exemplified by the adaptation
of CHROMagar Orientation for the detection of Enterobacteriaceae with acquired
resistance to colistin (213). Where MALDI-TOF MS is available, the potential attri-
butes of chromogenic media should now be assessed in this context, as MALDI-TOF
MS may compensate for any lack of specificity and contribute to an even shorter
time to result.
When looking to the future, it is tempting to anticipate the systematic replacement
of culture methods with PCR-based methods to overcome the inherent delay associ-
ated with culture. However, there are also inherent drawbacks in the exclusive use of
molecular methods. For example, isolation of bacterial pathogens remains essential in
order to perform antimicrobial susceptibility testing, which means that effective culture
methods must still be available for investigation of samples that are positive using PCR.
In the long term, this issue may be resolved by whole-genome sequencing of species
directly from clinical samples. However, for most bacterial species there is currently
insufficient evidence to support the use of whole-genome sequencing to infer antimi-
crobial susceptibility even when a pure culture is available (214). Another inherent
disadvantage of PCR methods is that new genes or gene variants that may compromise
assay performance continuously appear. For example, a pathogen that harbors a new
gene conferring antimicrobial resistance may evade detection by PCR but is likely to be
recovered on chromogenic media due to phenotypic expression of resistance (200). For
some applications, the convenience offered by molecular methods is compelling, for
example, the ability to screen simultaneously and effectively for a wide variety of
enteric pathogens. For some other applications, the evidence suggests that chromo-
genic media are at least as convenient and sensitive as well as being more cost-
effective. As clinical bacteriology grows in complexity, not least due to the widespread
emergence of bacteria with resistance to multiple antimicrobials, chromogenic media
should no longer be assessed as individual tools but as potentially useful components
within diagnostic algorithms.
ACKNOWLEDGMENTS
The Freeman Hospital Microbiology Department has received funding from bio-
Mérieux for the development and evaluation of culture media, and I have performed
consultancy work for the same company. The Freeman Hospital Microbiology Depart-
ment has also received funding for evaluation studies from Bio-Rad Laboratories and
International Diagnostics Group.

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John D. Perry, Ph.D., D.Sc., is a Clinical Scien-

tist at the Freeman Hospital in Newcastle upon
Tyne, UK. His graduate and postgraduate train-
ing were at Northumbria University, where he
serves as a Visiting Professor. Dr. Perry has a
strong interest in the laboratory aspects of anti-
microbial chemotherapy and serves as a senior
editor for the Journal of Antimicrobial Chemo-
therapy. He also has a longstanding interest in
microbial biochemistry and the design and
evaluation of new culture media and other di-
agnostic methods through the application of enzyme substrates and novel
growth inhibitors.

AUTHOR CORRECTION
crossm
Correction for Perry, “A Decade of

Development of Chromogenic Culture
Media for Clinical Microbiology in an Era
of Molecular Diagnostics”
John D. Perry
Microbiology Department, Freeman Hospital, Newcastle upon Tyne, UK
Volume 30, no. 2, p. 449 – 479, 2017, https://doi.org/10.1128/CMR.00097-16. Page

457, line 1: “a simple disk test for pyroglutamyl aminopeptidase” should read “a simple Published 13 September 2017
disk test for L-proline aminopeptidase.” Citation Perry JD. 2017. Correction for Perry,
“A decade of development of chromogenic
culture media for clinical microbiology in an
era of molecular diagnostics.” Clin Microbiol Rev
30:vii. https://doi.org/10.1128/CMR.00073-17.
© Crown copyright 2017. The government of
Australia, Canada, or the UK (“the Crown”) owns
the copyright interests of authors who are
government employees. The Crown Copyright
is not transferable.
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SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 449 Published 25 January 2017

April 2017 Volume 30 Issue 2 Clinical Microbiology Reviews cmr.asm.org 449

KEYWORDS carbapenemase-producing Enterobacteriaceae, chromogenic media,

C hromogenic media utilize synthetic chromogenic enzyme substrates in order to

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CHROMOGENIC MEDIA FOR DETECTION OF SPECIFIC (NONENTERIC)

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TABLE 1 Timeline of the evolution of chromogenic culture media applied to clinical

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chromID Pseudomonas (11). The medium is notable as it is the ﬁrst chromogenic

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Streptococcus agalactiae (Group B Streptococcus)

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Craven et al., 250/81 Vaginal, rectal chromID Strepto B 87.7 97.6

Louie et al., 1,025/243 Vaginal, rectal CNA blood agar 82.7

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Poisson et al., 285/84 Vaginal, rectal Broth (CN-TH), CHROMagar StrepB 79 92 96 95

Kwatra et al., 260/92 Vaginal, rectal CHROMagar StrepB 85.9 88

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Urinary Tract Pathogens

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feasible alternative to conventional media for isolation and identiﬁcation of most

CHROMOGENIC MEDIA FOR DETECTION OF ENTERIC PATHOGENS

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Carson et al., 2013 (69) 50/47 None chromID C. difﬁcile 100

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␤-ribosidase has provided one alternative means of detection (85). HardyCHROM SS is

Shiga Toxin-Producing Escherichia coli

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detected by culture on CHROMagar STEC, and a further 68 false-positive colonies were

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CHROMOGENIC MEDIA FOR DETECTION OF ANTIMICROBIAL-RESISTANT

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Methicillin-Resistant Staphylococcus aureus

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Dodémont et al., 1,220/107 Nasal, throat, perineal, and Direct inoculation

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large study that compared ﬁve chromogenic agars with bile-esculin-azide-vancomycin

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Paniagua et al., 500/41 Stool (500) chromID ESBL 100 63

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Blane et al., 298/116 Stool (298) chromID ESBL 63 75

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Papadimitriou-Olivgeris 177/86 chromID CARBA 96.5 91.2 Greece KPC

Davies et al., 2016 236/33 Brilliance CRE 97 87.9 UK NDM

Papadimitriou-Olivgeris 912/329 Brilliance CRE 96.8 90.9 Greece KPC

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